RNA polymerase mediated transcription, also referred to herein as "RNA polymerase mediated amplification," is based on the ability of an RNA polymerase to use a nucleic acid template to catalyze ribonucleic acid synthesis. The RNA polymerase synthesizes ribonucleic acid transcripts complementary to the template.
RNA polymerase mediated amplification is initiated by the binding of an RNA polymerase to a promoter region which is usually double-stranded. The RNA polymerase proceeds downstream from the promoter region and synthesizes ribonucleic acid in a 5' to 3' direction. Multiple copies, for example, in the range of 100-3,000, of RNA transcripts are produced by RNA polymerase mediated amplification using a single template.
Factors affecting RNA polymerase mediated amplification efficiency are known to those of ordinary skill in the art. E.g., see, Milligan et al., Nucleic Acids Research 15:8783-8798, 1987 ("Milligan et al.") (hereby incorporated by reference herein). Using a linear template most, but not all, of the promoter sequence needs to be part of a double-stranded promoter region for RNA polymerase mediated amplification. For example, Milligan et al. refers to a "bottom strand" which contains the promoter sequence upstream from the template and a "top strand" which is nucleic acid complementary to the bottom strand. Milligan et al. indicates that for T7 RNA polymerase, the top strand can be several nucleotides shorter on either the 3' or 5' terminus of the promoter sequence. According to Milligan et al. at pages 8791-8792:
As the top strand is shortened from its 3' end, neither the site of initiation nor the amount of products is significantly altered until nucleotide -3 is removed. Further elimination of nucleotides causes a decrease in the amount of full length product, but not any change in the point of initiation, until the nucleotide at -8 is eliminated at which point there are no detectable full length products. A similar, less complete, experiment removing nucleotides from the 5' terminus of the top strand shows no change in the transcription products or yields until nucleotide -14 is removed. Thus, the top strand does not have to cover the entire T7 consensus promoter, but can be up to 3 nucleotides shorter on either the 3' or 5' terminus. PA1 . . predicated on the use of an oligonucleotide probe, suitable for hybridization with a segment of a target nucleic acid sequence, that has linked thereto a moiety that is capable of initiating the production of a plurality of RNA transcripts, themselves containing sequence operable for their multiple self-replication. PA1 Specific nucleic acid sequences are amplified through the use of transcribable hairpin probes. The probe comprises a single stranded self-complementary sequence which, under hybridizing conditions, forms a hairpin structure having a functional promoter region, and further comprises a transcribable sequence extending from the '5 end of the hairpin sequence and a probe sequence which may be comprised in the transcribable 5' sequence or in a sequence extending from the 3' end of the hairpin sequence. PA1 This invention involves the use of a nucleic acid hybridization probe comprising at least the following essentials: a probe sequence of approximately 15-115 nucleotides in length surrounded on both sides by complementary nucleic acid sequences which are considerably shorter than the probe sequence, preferably not greatly in excess of one-half the length of the probe sequence. This combination of three sequences forms a simple molecular allosteric switch. PA1 a stem made up of a first and a second nucleic acid sequence which are substantially complementary to each other, PA1 and a single-stranded loop region located between the first and second nucleic acid sequences, wherein an RNA polymerase promoter sequence is located substantially within the loop region.
Milligan et al. also indicates that removal of additional top strand bases results in decreasing activity until nucleotide -8 is removed, and removal of nucleotides to -14 resulted in no full length transcription products being observed. Other RNA polymerases, such as T3 RNA polymerase, have similar properties in that a double-stranded promoter is generally needed for transcription activity, however, the promoter sequence need not be completely double-stranded.
Daubendiek et al., J. Am. Chem. Soc. 117:7818-7819, 1995 ("Daubendiek et al.") (hereby incorporated by reference herein), indicates that a circular oligonucleotide can serve as a template for T7 polymerase. At page 7818, first column, second paragraph, Daubendiek et al., notes that transcription was said to occur " . . . in the absence of RNA primers, in the absence of RNA promoter sequences, and in the absence of any duplex structure at all."
Transcription templates include RNA and DNA, and may be single-stranded or double-stranded. Kacian et al., U.S. Pat. No. 5,399,491, and Burg et al., U.S. Pat. No. 5,437,990, both illustrate RNA polymerase mediated amplification using a DNA template (both of these references are hereby incorporated by reference herein). Kacian et al., International Publication No. PCT/US93/04015, International Publication No. WO 93/22461, illustrates transcription using an RNA template (hereby incorporated by reference herein).
Transcription, along with other techniques which amplify nucleic acid using a nucleic acid template such as the polymerase chain reaction method (PCR), the ligase chain reaction (LCR), and Q.beta. replicase are particularly useful as part of a diagnostic technique where amplified nucleic acid is detected to indicate the presence of a target sequence.
Publications mentioning transcription as part of a diagnostic technique include Kacian et al., U.S. Pat. No. 5,399,491; Burg et al., U.S. Pat. No. 5,437,990; Kacian et al., International Publication No. PCT/US93/04015, International Publication No. WO 93/22461; Gingeras et al., International Application No. PCT/US87/01966, International Publication No. WO 88/01302; Gingeras et al., International Application No. PCT/US88/02108, International Publication No. WO 88/10315; Davey and Malek, EPO Application No. 88113948.9, European Publication No. 0 329 822 A2; Malek et al., U.S. Pat. No. 5,130,238; Urdea, International Application No. PCT/US91/00213, International Publication No. WO 91/10746; McDonough et al., International Application No. PCT/US93/07138, International Publication No. WO 94/03472; Kacian et al., International Publication No. PCT/US93/04015, International Publication No. WO 93/22461; and Ryder et al., International Application No. PCT/US94/08307, International Publication Number WO 95/03430.
Different schemes can be employed to bring about transcription in the presence of target sequence. For example, Axelrod et al., International Application No. PCT/US89/03884, International Publication No. WO 90/02820, on page 6, lines 29-35, mentions an "invention":
Dattagupta, European Patent Application No. 90120652.4, Publication Number 0 427 074 A2, in the abstract asserts:
Dattagupta, U.S. Pat. No. 5,215,899, describes the same type of technology as the Dattagupta European patent application.
Lizardi et al., International Application No. PCT/US89/04275, International Publication No. WO 90/03446 ("Lizardi et al.,") , describes nucleic acid probes containing "molecular switches." The molecular switches described by Lizardi et al. are closed in the absence of target sequence, but open in the presence of target sequence. According to Lizardi et al. at page 10, lines 13-23:
None of the references described herein are admitted to be prior art to the claimed invention.